Stolonoxides

ABSTRACT

Naturally occuring stolonoxide compounds, and derivatives thereof, have antitumour activity. Typical compounds are of the formula (I) or a derivative thereof.

[0001] The present invention relates to stolonoxides, includingstolonoxide minor metabolites from the ascidian Stolonica socialis andderivatives thereof, and further relates to the cytotoxicity ofstolonoxides.

BACKGROUND OF THE INVENTION

[0002] Marine ascidians continue to focus interest of both marinechemical and biomedical research. The chemistry of ascidians isdominated by amino acid derived compounds either peptides or alkaloids.However the non nitrogenous metabolites from ascidians, althoughconsiderably minor in number, are by no means less important, seeFaulkner, D. J. Nat. Prod. Rep. 2000, 17, 7-55, and references citedtherein. Among these non nitrogenous metabolites oxylipins, metabolitesderived by biooxidation of fatty acids, are rarely encountered, seeGerwick, W. H. Lipids 1996, 31, 1215-1231.

[0003] The isolation of stolonoxide A, a novel cyclic peroxide isolatedas its methyl ester from a sample collected from Tarifa Island (Cádiz,Spain) Stolonica socialis, has been reported, see Fontana, A; González,M. C.; Gavagnin, M.; Templado, J.; Cimino, G. Tetrahedron Lett. 2000,41, 429-4-32.

SUMMARY OF INVENTION

[0004] According to the present invention, we provide pharmaceuticalcompositions of stolonoxide compounds. We also provide new stolonoxidecompounds, including derivatives of naturally occuring compounds.

[0005] In one aspect, we provide pharmaceutical compositions ofstolonoxide compounds of the formula:

[0006] and derivatives thereof.

[0007] The compounds include natural isolates and compounds synthesisedtherefrom, and have cytotoxic activity against tumour cell lines.

[0008] Thus, the present invention provides a method of treating anymammal, notably a human, affected by cancer which comprisesadministering to the affected individual a therapeutically effectiveamount of a compound of the invention, or a pharmaceutical compositionthereof.

[0009] The present invention also relates to pharmaceutical preparationsincluding a pharmaceutically acceptable carrier and which contain as anactive ingredient a compound or compounds of the invention. Theinvention extends to the processes for the preparation of thepharmaceutical compositions. Certain of the compounds can be readilyisolated as mixtures, and such mixtures may be used in the compositions.

[0010] The present invention also extends to the compounds of theinvention for use in a method of treatment, and to the use of thecompounds in the preparation of a composition for treatment of cancer.

[0011] We further provide new stolonoxide compounds and derivatives,with the exception of the known methyl ester of stolonoxide A offormula:

PREFERRED EMBODIMENTS

[0012] Preferred stolonoxides of this invention are stolonoxides A, B, Cand D of the formulae:

[0013] where R is H for the parent stolonoxide compound. Derivatives ofthe stolonoxides of this invention include those compounds formed byderivatisation of the carboxylic acid, which ate thus compounds where Ris not hydrogen.

[0014] Examples of derivatives which form part of this invention includesalts, especially alkali metal salts such as the sodium salt; esters,especially optionally substituted aliphatic or aryl esters such as C₁-C₆or higher alkyl esters, cycloalkyl esters, alkenyl esters, alkynylesters, aryl esters such as phenyl esters, aralkyl esters, aralkenylesters, aralkynyl esters, all of which may be optionally substitutedwith alkyl alkoxy, alkylthio, halogen, haloalkyl, carboxyl, hydroxy,hydroxyalkyl, aryl or aminoalkyl groups, and where the aryl groups maybe heterocyclic; or amides, especially simple amides or N-substitutedamides.

[0015] Suitable halogen substituents in the compounds of the presentinvention include F, Cl, Br and I.

[0016] Alkyl groups preferably have from 1 to 12 carbon atoms, morepreferably 1 to 8 carbon atoms, still more preferably 1 to 6 carbonatoms, and most preferably 1, 2, 3 or 4 carbon atoms. Methyl, ethyl andpropyl including isopropyl are particularly preferred alkyl groups inthe compounds of the present invention. As used herein, the term alkyl,unless otherwise modified, refers to both cyclic and noncyclic groups,although cyclic groups will comprise at least three carbon ring members.

[0017] Preferred alkenyl and alkynyl groups in the compounds of thepresent invention have one or more unsaturated linkages and from 2 to 12carbon atoms, more preferably 2 to 8 carbon atoms, still moreprefereably 2 to 6 carbon atoms, even more prefereably 2, 3 or 4 carbonatoms. The terms alkenyl and alkynyl as used herein refere to bothcyclic and noncyclic groups, although straight or branched noncyclicgroups are generally more preferred.

[0018] Preferred alkoxy groups in the compounds of the present inventioninclude groups having one or more oxygem linkages and from 1 to 12carbon atoms, more preferably from 1 to 8 carbon atoms, and still morepreferably 1 to 6 carbon atoms, and most preferably 1, 2, 3 or 4 carbonatoms.

[0019] Preferred alkylthio groups in the compounds of the presentinvention have one or more thioether linkages and from 1 to about 12carbon atoms, more prefereably from 1 to about 8 carbon atoms, and stillmore preferably 1 to about 6 carbon atoms. Alkylthio groups having 1, 2,3 or 4 carbon atoms are particularly preferred.

[0020] Preferred aminoalkyl groups include those groups having one ormore primary, secondary and/or tertiary amine groups, and from 1 to 12carbon atoms, more preferably 1 to 8 carbon atoms, still more preferably1 to 6 carbon atoms, even more preferably 1, 2, 3 or 4 carbon atoms.Secondary and tertiary amine groups are generally more preferred thanprimary amine moieties.

[0021] Preferred amido groups include primary, secondary or tertiaryamido groups, and include secondary or tertiary amido groups having oneor more alkyl groups.

[0022] Suitable heterocyclic groups in the compounds of the presentinvention preferably contain one, two or three heteroatoms selected fromnitrogen, oxygen and sulphur atoms and include heteroaromatic groupssuch as, for example, coumarinyl including 8-coumarinyl, quinolinylincluding 8-quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl,thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl andbenzothiazol; and heteroalicyclic groups such as, for example,tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino andpyrrolindinyl groups.

[0023] Suitable carbocyclic aryl groups in the compounds of the presentinvention include single and multiple ring compounds, including multiplering compounds that contain separate and/or fused aryl groups. Typicalcarbocyclic aryl groups contain 1 to 3 separate or fused rings and from6 to about 18 carbon ring atoms. Specifically preferred carbocyclicarykl groups include phenyl including substituted phenyl, such as2-substituted phenyl, 3-substituted phenyl, 2,3-substituted phenyl,2,5-substituted phenyl, 2,3,5-substituted and 2,4,5-substituted phenyl,including where one or more of the phenyl substituents is anelectron-withdrawing group such as halogen, cyano, nitro, alkanoyl,sulfinyl, sulfonyl and the like; naphthyl including 1-naphthyl and2-naphthyl; biphenyl; phenanthryl; and anthracyl.

[0024] References herein to substituted groups in the compounds of thepresent invention refer to the specified moiety that may be substitutedat one or more available positions by one or more suitable groups, forexample, halogen such as fluoro, chloro, bromo and iodide; cyano;hydroxyl; nitro; azido; alkanoyl such as a C₁-C₆ alkanoyl group such asacyl and the like; carboxamido; alkyl groups including those groupshaving 1 to 2 carbon atoms or from 1 to 6 carbon atoms and morepreferably 1-3 carbon atoms; alkenyl and alkynyl groups including groupshaving one or more unsaturated linkages and from 2 to 12 carbon or from2 to 6 carbon atoms; alkoxy groups having those having one or moreoxygen linkages and from 1 to 12 carbon atoms or 1 to 6 carbon atoms;aryloxy such as phenoxy; alkylthio groups including those moietieshaving one or more thioether linkages and from 1 to 12 carbon atoms orfrom 1 to 6 carbo atoms; alkylsulfinyl groups including those moietieshaving one or more sulfinyl linkages and from 1 to 12 carbon atoms orfrom 1 to 6 carbon atoms; alkylsulfinyl groups including those moietieshaving one or more sulfonyl linkages and from 1 to 12 carbon atoms orfrom 1 to 6 carbon atoms; aminoalkyl groups such as groups having one ormore N atoms and from 1 to 12 carbon atoms or from 1 to 6 carbon atoms;carbocyclic aryl having 6 or more carbons, particularly phenyl (forinstance a substituted or unsubstituted biphenyl moiety); and aralkylsuch as benzyl.

[0025] The sidechain in the compounds can be 10-α or 10-β. The Δ^(19,20)double bond in the compounds can be cis or trans.

[0026] Examples of derivatives include compounds of formula (1a), (2a),(3a) and (4a), where the suffix “a” indicates a sodium salt, andcompounds of formula (1b), (2b), (3b) and (4b) where the suffix “b”indicates a methyl ester. The compounds 2, 2a, 2b, 3a, and 4a arepreferred compounds per se of this invention, while preferred compoundsfor antitumor activity include these compounds and compounds 1, 1a and1b. The compounds of this invention can be in substantially pure form,and can be free from other cells or material from Stolonica socialis.

[0027] Examples of pharmaceutical compositions include any solid(tablets, pills, capsules, granules, etc.) or liquid (solutions,suspensions or emulsions) with suitable composition or oral, topical orparenteral administration, and they may contain the pure compound or incombination with any carrier or other pharmacologically activecompounds. These compositions may need to be sterile when administeredparenterally.

[0028] Administration of the compounds or compositions of the presentinvention may be by any suitable method, such as intravenous infusion,oral preparations, intraperitoneal and intravenous administration. Weprefer that infusion times of up to 24 hours are used, more preferably2-12 hs, with 2-6 hours most preferred. Short infusion times which allowtreatment to be carried out without an overnight stay in hospital areespecially desirable. However, infusion may be 12 to 24 hours or evenlonger if required. Infusion may be carried out at suitable intervals ofsay 2 to 4 weeks. Pharmaceutical compositions containing compounds ofthe invention may be delivered by liposome or nanosphere encapsulation,in sustained release formulations or by other standard delivery means.

[0029] The correct dosage of the compounds will vary according to theparticular formulation, the mode of application, and the particularsitus, host and tumour being treated. Other factors like age, bodyweight, sex, diet, time of administration, rate of excretion, conditionof the host, drug combinations, reaction sensitivities and severity ofthe disease shall be taken into account. Administration can be carriedout continuously or periodically within the maximum tolerated dose.

[0030] The compounds and compositions of this invention may be used withother drugs to provide a combination therapy. The other drugs may formpart of the same composition, or be provided as a separate compositionfor administration at the same time or a different time.

[0031] The identity of the other drug is not particularly limited, andsuitable candidates included:

[0032] a) drugs with antimitotic effects, especially those which targetcytoskeletal elements, including microtubule modulators such as taxanedrugs (such as taxol, paclitaxel, taxotere, docetaxel), podophylotoxinsor vinca alkaloids (vincristine, vinblastine);

[0033] b) antimetabolite drugs such as 5-fluorouracil, cytarabine,gemcitabine, purine analogues such as pentostatin, methotrexate);

[0034] c) alkylating agents such as nitrogen mustards (such ascyclophosphamide or ifosphamide);

[0035] d) drugs which target DNA such as the antracycline drugsadriamycin, doxorubicin, pharmorubicin or epirubicin;

[0036] e) drugs which target topoisomerases such as etoposide;

[0037] f) hormones and hormone agonists or antagonists such asestrogents, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyprotrone or octreotide;

[0038] g) drugs which target signal transduction in tumour cellsincluding antibody derivatives such as herceptin;

[0039] h) alkylating drugs such as platinum drugs (cis-platin,carbonplatin, oxaliplatin, paraplatin) or nitrosoureas;

[0040] i) drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors;

[0041] j) gene therapy and antisense agents;

[0042] k) antibody therapeutics;

[0043] l) other bioactive compounds of marine origin, notably thedidemnins such as aplidine or the ecteinascidins such as Et-743;

[0044] m) steroid analogues, in particular dexamethasone;

[0045] n) anti-inflammatory drugs, in particular dexamethasone; and

[0046] o) anti-emetic drugs, in particular dexamethason.

[0047] Antitumour Assays

[0048] Cell Cultures. Cells were maintained in logarithmic phase ofgrowth in Eagle's Minimum Essential Medium, with Earle's Balanced Salts,with 2.0 mM L-glutamine, with non-essential amino acids, without sodiumbicarbonate (EMEM/neaa); supplemented with 10% Fetal Calf Serum (FCS),10⁻² M sodium bicarbonate and 0.1 g/l penicillin-G+streptomycin sulfate.

[0049] A simple screening procedure has been carried out to determineand compare the antitumour activity of these compounds, using an adaptedform of the method described by Bergeron et al (1984). The tumour cellline employed were P-388 (suspension culture of a lymphoid neoplasm fromDBA/2 mouse), A-549 (monolayer culture of a human lung carcinoma), HT-29(monolayer culture of a human colon carcinoma) and MEL-28 (monolayerculture of a human melanoma).

[0050] P-388 cell were seeded into 16 mm wells at 1×10⁴ cells per wellin 1 ml aliquots of MEM 5FCS containing the indicated concentration ofdrug. A separate set of cultures without drug was seeded as controlgrowth to ensure that cells remained in exponential phase of growth. Alldeterminations were carried out in duplicate. After three days ofincubation at 37° C., 10% CO₂ in a 98% humid atmosphere, an approximateIC₅₀ was determined by comparing the growth in wells with drug to thegrowth in wells contol.

[0051] A-549, HT-29 and MEL-28 were seeded into 16 mm wells at 2×10⁴cells per well in 1 ml aliquots of MEM 10FCS containing the indicatedconcentration of drug. A separate set of cultures without drug wasseeded as control growth to ensure that cells remained in exponentialphase of growth. All determinations were carried out in duplicate. Afterthree days of incubation at 37° C., 10% CO₂ in a 98% humid atmosphere,the wells were stained with 0.1% Crystal Violet. An approximate IC₅₀ wasdetermined by comparing the growth in wells with drug to the growth inwells control.

[0052] Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J. Kline.Robert G. Hughes, Jr., Gary T. Elliot and Carl W. Porter. Antineoplasticand antiherpetic activity of spermidine catecholamide iron chelators.Biochem. Bioph. Res. Comm. 1984, 121(3), 848-854.

[0053] Alan C. Schroeder, Robert G. Hughes, Jr. and Alexander Bloch.Effects of Acyclic Pyrimidine Nucleoside Analoges. J. Med. Chem. 1981,24 1078-1083. Antitumoral in vitro data IC₅₀μg/ml Compound P 388 A 549HT 29 MEL 28 Mixture 1 and 2 (9:1) 0.01 0.1 0.1 0.1 Mixture 1a and 2a(6:4) 0.05 0.1 0.1 0.1 Mixture 3a and 4a (6:4) 0.01 0.01 0.05 0.1 Methylester (2b) 0.5 0.1 0.1 0.5 Methyl ester (1b) 0.1 0.1 0.1 Not tested

EXAMPLES OF THE INVENTION

[0054] Bioassay guided isolation of the metabolites of the ascidianStolonica socialis from Tarifa Island (Cádiz, Spain) led to a series ofcyclic peroxides. A 9:1 mixture of stolonoxide A (1) and the newstolonoxide B (2), a 6:4 mixture of their corresponding sodium salts(1a, 2a), and a minor 6:4 mixture of the new stolonoxides C (3) and D(4) isolated as their sodium salts (3a) and (4a). Their structures wereestablished by spectroscopic study of both the natural mixtures, whoseconstituents differ in the geometry of a double bond, and thecorresponding methyl esters.

[0055] As a part of the research project carried aimed to examine thebiomedical potential of new metabolites from ascidians of the southerncoast of Spain, see (a) Durán, R.; Zubía, E.; Ortega, M. J.; Naranjo,S.; Salvá, J. Tetrahedron 1999, 55, 13225-1-13232; (b) Ortega, M. J.;Zubía, E.; Ocaña, J. M.; Naranjo, S.; Salvá, J. Tetrahedron accepted forpublication, we collected specimens of the ascidian Stolonica socialis(Hartmeyer, 1903) belonging to the Styelidae Family. Specimens of S.socialis were collected by hand using SCUBA off Tarifa Island (Cádiz,Spain) and immediately frozen.

[0056] The frozen material was extracted with an acetone:methanolmixture (1:1). After evaporation of the solvent the aqueous residue wasextracted with Et₂O to yield a cytotoxic extract against the tumor celllines of mouse lymphoma P-388, human lung carcinoma A-549 and humancolon carcinoma HT-29 (IC₅₀=5 μg/mL). Column chromatography of the Et₂Osoluble material yielded six fractions of which fraction E was selectedguided by the cytotoxicity against the tumor cell lines mentioned(IC₅₀=0.05 μg/mL). Further purification of this fraction using reversedphase HPLC allowed isolation of three pairs of compounds: a 9:1 mixtureof stolonoxide A (1) and stolonoxide B (2), a 6:4 mixture of theircorresponding sodium salts (1a and 2a), and a 6:4 mixture of the sodiumsalts of stolonoxide C and stolonoxide D (3 and 4). The individualcomponents of each of these three pairs of compounds proved to beunseparable under all the HPLC conditions assayed and therefore it wasnecessary to derivatise the natural constituents.

[0057] Experimental Section

[0058] General. Optical rotations were measured on a Perkin-Elmer 241polarimeter. IR and UV spectra were recorded on a Genesis Series FT IRMattson and Phillips PU 8710 spectrophotometer, respectively. ¹H and ¹³CNMR spectra were recorded at 400 and 100 MHz, respectively, on a VarianUnity 400 spectrometer using CDCl₃ as solvent. Proton chemical shiftswere referenced to the residual CDCl₃ signal at δ 7.26 and ¹³C NMRspectra were referenced to the central peak of CDCl₃ at δ 77.0.^(′)H-¹H-COSY, LR COSY, HMQC and HMBC were performed using standardVARIAN pulse sequences. Assignments marked with an asterisk may beinterchanged. Mass spectra were recorded on a VG Autospec spectrometer.Column chromatography was carried out using Merck Silica gel 60 (70-230mesh). HPLC separations were performed on a LaChrom-Hitachi apparatusequipped with LiChrosorb RP-18 and LiChrosorb Si 60 columns using adifferential refractometer. All solvents were spectral grade or weredistilled from glass prior to use.

[0059] Collection, Extraction, and Isolation Procedures. Specimens ofStoloilica socialis (76 g dry weight) were collected by hand using SCUBAoff Tarifa Island in May 1996 and immediately frozen. The frozen tissuewas extracted with acetone-methanol (1:1) at room temperature. Thefiltered solution was evaporated under reduced pressure yielding anaqueous residue that was further extracted sequentially with Et₂O (4×450mL) and n-BuOH (3×500 mL). The Et₂O extract was filtered andconcentrated to yield 5.7 g of an orange cytotoxic oil (5.7 g) which waschromatographed on a SIO₂ column eluting with mixtures of increasingpolarities from hexane to Et₂O and, subsequently, EOAc, CHCl₃/MeOH (1:1)and MeOH. Selected fractions were subjected to reversed phase HPLCseparations on a preparative LiChrosorb RP-18 column eluting withMeOH/H₂O (9:1) to afford in order of elution: stolonoxide C and D sodiumsalts (3 and 4, 17 mg, 0.220% dry wt), stolonoxide A and B sodium salts(1a and 2a, 58 mg, 0.076% dry wt), stolonoxide A and B (1 and 2, 80 mg,0.105% dry wt). Final purification was accomplished by HPLC on reversedphase mode using mixtures of MeOH/H₂O. TABLE 1 ¹H NMR data recorded inCDC1₃ for the natural metabolites from Stolonica socialis ^(a) 1^(b)2^(b) 1a 2b 3 4 2 2.49 (dd, 15.8, 7.2) 2.26 (m) 2.34 (m) 2.44 (dd, 15.8,5.3) 3 4.52 (m) 4.45 (m) 4.48 (m) 4 1.95 (m, Heq) 1.90 (m, Heq) 1.91 (m,Heq) 1.60 (m, Hax) 1.49 (m, Hax) 1.52 (m, Hax) 5 1.73 (m) 1.60 (m) 1.65(m) 6 4.07 (m) 4.00 (m) 4.02 (m) 7 3.87 (m) 3.79 (m) 3.82 (bd, 6.0) 81.95 (m) 1.92 (m) 1.95(m) 1.77 (m) 1.66 (m) 1.70 (m) 9 2.00 (m) 1.97 (m)1.97 (m) 1.44 (m) 1.43 (m) 1.43 (m) 10 3.87 (m) 3.88 (m) 3.88 (m) 111.58 (m) 1.60 (m) 1.60 (m) 1.33 (m) 1.32 (m) 1.34 (m) 12 1.30-1.20 (m)1.30-1.20 (m) 1.30-1.20 (m) 13 1.30-1.20 (m) 1.30-1.20 (m) 1.30-1.20 (m)14 1.30-1.20 (m) 1.30-1.20 (m) 1.30-1.20 (m) 15 1.30-1.20 (m) 1.30-1.20(m) 1.30-1.20 (m) 16 2.15 (m) 2.14 (m) 2.15(m) 17 5.44 (m) 5.30 (m) 5.44(m) 5.30 (m) 5.44 (m) 5.31 (dt, 10.9, 7.4) 18 6.25 (m) 5.96 (bt, 10.8)6.24 (m) 5.94 (bt, 10.6 6.24 (m) 5.93 (bt, 10.9) 19 6.25 (m) 6.30 (m)6.24 (m) 6.31 (m) 6.24 (m) 6.31 (bdd, 15.2, 10.9) 20 5.44 (m) 5.65 (m)5.44 (m) 5.65 (m) 5.44 (m) 5.65 (bdt, 15.2, 6.6) 21 2.27 (bdd, 14.7,7.5) 2.17 (m) 2.26 (m) 2.17 (m) 2.27 (bdd, 14.8, 7.5) 2.18 (m) 22 2.13(m) 2.14 (m) 2.15 (m) 23 5.82 5.82 (ddt, 16.9, 10.3, 6.5) 5.82 (ddt,17.0, 10.2, 6.5) (ddt, 17.0, 10.2, 6.5) 24 5.03 5.03 bd, 16.9) 5.03 (bd,17.0) (ddd, 17.0, 1.7, 1.6) 4.96 (bd, 10.2) 4.97 (bd, 10.3) 4.96 (bd,10.2)

[0060] TABLE 2 ¹³C NMR data recorded in CDCI₃ for the naturalmetabolites from Stolonica socialis ^(a) 1^(b) 2^(b) 1a 2b 3 4 1 175.1(s) 177.1 (s) 176.0 (s) 2  38.4 (t)  41.3 (t)  40.0 (t) 3  77.4 (d) 79.2 (d)  78.5 (d) 4  29.0 (t)  29.0 (t)  29.0 (t) 5  25.1 (t)  25.1(t)  25.1 (t) 6  83.8 (d)  84.0 (d)  84.0 (d) 7  78.5 (d)  78.7 (d) 78.6 (d) 8  27.7 (t)  28.1 (t)  28.0 (t) 9  31.6 (t)  31.6 (t)  31.6(t) 10  80.0 (d)  79.9 (d)  79.9 (d) 11  35.5 (t)  35.6 (t)  35.6 (t) 12 26.0 (t)  25.9 (t)  26.0 (t) 13  29.6 (t)^(c)  29.7 (t)^(c)  29.7(t)^(c) 14  29.5 (t)^(c)  29.6 (t)^(c)  29.6 (t)^(c) 15  29.2 (t)^(c) 29.3 (t)^(c)  29.3 (t)^(c) 16  27.4 (t)  27.7  27.5 (t)  27.7 (t)  27.5(t)  27.8 (t) (t) 17 132.4 130.4 132.3 130.4 132.4 130.4 18 123.4 128.4123.5 128.5 123.5 128.5 19 124.0 126.0 124.0 126.0 124.0 126.1 20 130.8133.4 130.8 133.5 130.8 133.5 21  26.8 (t)  32.2  26.9 (t)  32.2 (t) 26.9 (t)  32.2 (t) (t) 22  33.7 (t)  33.7 (t)  33.7 (t) 23 138.2 (d)138.2 (d) 138.2 (d) 24 114.7 (t) 114.8 (t) 114.8 (t)

[0061] Stolonoxides A and B (1 and 2): amorphous powder; [α]_(D) ²⁵−62.90 (c 0.49, CHCl₃); IR (film) 3460, 1745, 1237, 1064 cm⁻¹; UV (MeOH)λ_(max)234 (ε=20400) nm; ₁H NMR (CDCl₃) see Table 1; ¹³C NMR (CDCl₃) seeTable 2; EIMS (70 eV) m/z (rel. int.) 406 (18), 388 (7), 344 (5), 261(10), 219 (5), 135 (15), 121 (23), 95 (50), 67 (100); HREIMS m/z406.2734, C₂₄H₃₈O₅ requires m/z 406.2719.

[0062] Stolonoxides A and B sodium salts (1a and 2a): amorphous powder;[α]_(D) ²⁵ −76.8° (c 0.19, CHCl₃); IR (film) 1579, 1435, 1070 cm¹; UV(MeOH) λ_(max) 234 (ε 19600) nm; ¹H NMR (CDCl₃) see Table 1; ¹³C NMR(CDCl₃) see Table 2; (−)LRESIMS m/z (rel. int.) 405 [M−Na]−(100),(+)LRESIMS m/z (rel. int.) 429 [M+H]⁺ (100), 451 [M+Na]⁺ (5); HREIMS m/z406.2766, C₂₄H₃₈O₅ requires m/z 406.2719.

[0063] Stolonoxides C and D sodium salts (3a and 4a): amorphous powder;[α]_(D) ²⁵ +38.0° (c 0.2, CHCl₃); IR (film) 1584, 1440, 1057 cm⁻¹; UV(MeOH) λ_(max) 234 (ε=22500) nm; ¹H NMR (CDCl₃) see Table 1; ¹³C NMR(CDCl₃) see Table 2; (−)LRESIMS m/z (rel. int.) 405 [M−Na]⁻ (100),(+)LRESIMS m/z (rel. int.) 429 [M+H]⁺ (100); HREIMS m/z 388.2639,C₂₄H₃₆O₄ requires m/z 388.2614.

[0064] Methylation of the mixture of stolonoxides A (1) and B (2):Excess of CH₂N₂ was added to a solution of the mixture of stolonoxide A(1) and B (2) (22 mg) in Et₂O (2 mL) at room temperature. After 1 h, thesolvent was removed under reduced pressure and the reaction crudesubjected to HPLC chromatography yielding stolonoxide A methyl ester(1b, 18 mg) and stolonoxide B methyl ester (2b, 2 mg).

[0065] Methylation of the mixtures 1a, 2a, and 3,4: Methylation of thesemixtures was performed using the same procedure described above but themixtures were previously acidified with a solution of 1N HCI.

[0066] Stolonoxide A methyl ester (1b): colorless oil; [α]_(D) ²⁵ −50.8°(c 0.39, CHCl₃); IR (film) 1745, 1640, 1200, 1060 cm⁻¹; UV (MeOH)λ_(max) 235 (ε=22000) nm- ¹H NMR (CDCl₃) 6.25 (m, 2H, H-18 and H-19),5.82 (ddt, J=16.9, 10.3, 6.6 Hz, 1H, H-23), 5.44 (m, 2H, H-17 and H-20),5.03 (ddd, J=16.9, 1.7, 1.6 Hz, 1H, H-24), 4.97 (bd, J=10.3 Hz, 1H,H-24), 4.54 (dddd, J=11.1, 7.7, 5.9, 2.2 Hz, 1H, H-3), 4.06 (ddd,J=11.1, 5.4, 2.4 Hz, 1H, H-6), 3.87 (m, 2H, H-7 and H-10), 3.69 (s, 3H,CH₃O—), 2.47 (dd, J=15.6, 7.7 Hz, 1H, H-2), 2.36 (dd, J=15.6, 5.9 Hz,1H, H-2), 2.28 (bdd, J=14.7, 7.4, 2H, H-21), 2.15 (m, 2H, H-16), 2.13(m, 2H, H-22), 1.99 (m, 1H, H-9), 1.95 (m, 1H, H-8), 1.92 (m, 1H,H-4eq), 1.77 (m, 2H, H-5ax and H-8), 1.70 (m, 1H, H-5eq), 1.58 (m, 1H,H-11),1.54 (m, 1H, H-4ax), 1.42 (m, 1H, H-9), 1.34 (m, 1H, H-11), 1.33(m, 1H, H-12), 1.30 (m, 6H, H-13, H-14 and H-15), 1.28 (in, 1H, H-12);¹³C NMR (CDCl₃) 170.4 (s, C-1), 138.2 (d, C-23), 132.4 (d, C-17), 130.8(d, C-20), 124.0 (d, C-19), 123.4 (d, C-18), 114.7 (t, C-24), 83.8 (d,C-6), 79.9 (d, C-10), 78.5 (d, C-7), 77.6 (d, C-3), 51.9 (q, CH₃O−),38.4 (t, C-2), 35.6 (t, C-11), 33.7 (t, C-22), 31.7 (t, C-9), 29.6 (t,C-13)*, 29.5 (t, C-14)*, 29.2 (t, C-15)*, 29.1 (t, C-4), 27.7 (t, C-8),27.5 (t, C-16), 26.8 (t, C-21), 26.0 (t, C-12), 25.2 (t, C-5); EIMS (70eV) m/z (rel. int.) 420 (2), 402 (18), 261 (25), 219 (7), 159 (15), 143(36), 135 (29), 121 (41), 67 (100); HREIMS m/z 420.2865, C₂₅H₄₀O₅requires m/z 420.2876.

[0067] Stolonoxide B methyl ester (2b): colorless oil; [α]_(D) ²⁵ −37.7°(c 0.26, CHCl₃); IR (film) 1745, 1200, 1090 cm⁻¹; UV (MeOH) λ_(max) 233(ε=19900) nm; ¹H NMR (CDCl₃) 6.32 (dd, J=15.1, 11.0 Hz, 1H, H-19), 5.94(t, J=11.0 Hz, 1H, H-18), 5.82 (ddt, J=16.9, 10.3, 6.6 Hz, 1H, H-23),5.65 (dt, J=15.1, 6.7 Hz, 1H, H-20), 5.31 (dt, J=11.0, 7.6 Hz, 1H,H-17), 5.03 (ddd, J=16.9, 1.6, 1.3 Hz, 1H, H-24), 4.97 (bd, J=10.3 Hz,1H, H-24), 4.54 (dddd, J=11.2, 7.7, 6.0, 2.3 Hz, 1H, H-3),4.06 (ddd,J=11.0, 5.6, 2.6 Hz, 1H, H-6), 3.87 (m, 2H, H-7 and H-10), 3.69 (s, 3H,CH₃O—), 2.47 (dd, J=15.6, 7.7 Hz, 1H, H-2), 2.36 (dd, J=15.6, 6.0 Hz,1H, H-2), 2.20 (m, 2H, H-21), 2.17 (m, 2H, H-22), 2.14 (m, 2H, H-16),1.99 (m, 1H, H-9), 1.95 (m, 1H, H-8), 1.92 (i, 1H, H4eq), 1.77 (m, 2H,H-5ax and H-8), 1.70 (m, 1H, H-5eq), 1.58 (m, 1H, H-11), 1.54 (m, 1H,H-4ax), 1.42 (m, 1H, H-9), 1.34 (m, 1H, H-11), 1.33 (m, 1H, H12), 1.30(m, 6H, H-13, H-14 and H-15), 1.28 (m, 1H, H-12); ¹³C NMR (CDCl₃) 170.4(s, C-1), 138.2 (d, C-23), 133.5 (d, C-20), 130.5 (d, C-17), 128.4 (d,C-18), 126.1 (d. C-19),114.7 (t, C-24), 83.8 (d, C-6), 79.9 (d, C-10),78.5 (d, C-7), 77.6 (d, C-3), 51.2 (q, CH₃O—), 38.4 (t, C-2), 35.6 (t,C-11), 33.6 (t, C-22), 32.7 (t, C-9), 32.2 (t, C-21), 29.6 (t, C-13)*,29.6 (t, C-14)*, 29.2 (t, C-15)*, 29.1 (t, C-4),27.7 (t, C-8), 27.7 (t,C-16),26.0 (t, C-12), 25.2 (t, C-5); EIMS (70 eV) m/z (rel. int.) 420(2), 402 (17), 261 (27), 219 (9), 159 (17), 143 (76), 121 (61), 67(100); HREIMS m)z 420.2866, C₂₅H₄₀O₅ requires m/z 420.2876.

[0068] Stolonoxides C and D methyl esters (3a and 4a): colorless oil;[α]_(D) ²⁵ +41.70° (c 0.3, CHCl₃); IR (film) 1745, 1200, 1060 cm⁻¹; UV(MeOH) λ_(max) 234 (ε=19600) nm; ¹H NMR (CDCl₃) see Table 1; ¹³C NMR(CDCl₃) see Table 2; EIMS (70 eV) m/z (rel. int.) 420 (2), 402 (23), 261(23), 219 (6), 159 (10), 143 (27), 121 (35), 67 (100); HREIMS m/z420.2863, C₂₅H₄₀O₅ requires m/z 420.2876.

[0069]3a: ′H NMR (CDCl₃) 6.25 (m, 2H, H-18 and H-19), 5.82 (ddt, J=17.0,10.2, 6.6 Hz, 1H, H-23), 5.44 (m, 2H, H-17 and H-20), 5.03 (bd, J=17.0Hz, 1H, H-24), 4.97 (bd, J=10.2 Hz, 1H, H-24), 4.54 (dddd, J=11.0, 7.7,5.9, 2.2 Hz, 1H, H-3), 4.03 (ddd, J=10.9, 6.0, 2.1 Hz, 1H, H-6), 3.86(m, 1H, H-10), 3.80 (q, J=6.0 Hz, 1H, H-7), 3.69 (s, 3H, CH₃O—), 2.47(dd, J=15.6, 7.7 Hz, 1H, H-2), 2.37 (dd, J=15.7, 5.9 Hz, 1H, H-2), 2.28(bdd, J=14.7, 7.4 Hz, 2H, H-21), 2.16 (m, 2H, H-16), 2.14 (m, 2H, H-22),2.00 (m, 1H, H-8), 1.98 (m, 1H, H-9), 1.94 (m, 1H, H-5eq), 1.92 (m, 1H,H-4eq), 1.80 (in, 1H, H-8), 1.65 (m, 1H, H-5ax), 1.56 (m, 1H, H-11),1.55 (m, 1H, H-4ax), 1.45 (m, 1H, H-9), 1.38 (m, 1H, H-12), 1.37 (m, 1H,H-11), 1.28 (m, 1H, H-12), 1.30-1.20 (m, 6H, H-13, H-14 and H-15); ¹³CNMR (CDCl₃) 170.4 (s, C-1), 138.2 (d, C-23), 132.4 (d, C-17), 130.9 (d,C-20), 124.0 (d, C-19), 123.5 (d, C-18), 114.8 (t, C-24), 83.7 (d, C-6),79.9 (d, C-10), 78.5 (d, C-7), 77.8 (d, C-3), 51.9 (q, CH₃O—), 38.4 (t,C-2), 35.6 (t, C11), 33.7 (t, C-22), 31.6 (t, C-9), 29.6 (t, C-13)*,29.5 (t, C-14)*, 29.2 (t, C-15)*, 28.9 (t, C-4), 28.0 (t, C-8), 27.5 (t,C-16), 26.9 (t, C-21), 26.1 (t, C-12), 25.7 (t, C-5).

[0070] 4a: ′H NMR (CDCl₃) 6.32 (m, 1H, H-19), 5.94 (bt, J=10.9 Hz, 1H,H-18), 5.82 (ddt, J=17.0, 10.2, 6.6 Hz, 1H, H-23), 5.66 (dt, J=14.4, 6.4Hz, 1H, H-20), 5.30 (dt, J=10.9, 7.3 Hz, 1H, H-17), 5.03 (bd, J=17.0 Hz,1H, H-24), 4.97 (bd, J=10.2 Hz, 1H, H-24), 4.54 (dddd, J=11.0, 7.7, 5.8,2.2 Hz, 1H, H-3), 4.03 (ddd, J=10.9, 6.0, 2.1 Hz, 1H, H-6), 3.86 (m, 1H,H-10), 3.80 (q, J=6.0 Hz, 1H, H-7), 3.69 (s, 3H, CH₃O—), 2.47 (dd,J=15.7, 7.7 Hz, 1H, H-2), 2.37 (dd, J=15.7, 5.8 Hz, 1H, H-2), 2.19 (m,2H, H-21), 2.14 (m, 2H, H-22), 2.13 (m, 2H, H-16), 2.00 (m, 1H,H-8),1.98 (m, 1H, H-9), 1.94 (m, 1H, H-5eq), 1.92 (m, 1H, H4eq), 1.80(m, 1H, H-8), 1.65 (m, 1H, H-5ax), 1.56 (m, 1H, H-11), 1.55 (m, 1H,H-4ax), 1.45 (m, 1H, H-9), 1.38 (m, 1H, H-12), 1.37 (m, 1H, H-11), 1.28(m, 1H, H-12), 1.30-1.20 (m, 6H, H-13, H-14 and H-15); ¹³C NMR (CDCl₃)170.4 (s, C-1), 138.2 (d, C-23),133.5 (d, C-20),130.4 (d, C-17),128.5(d, C-18), 126.0 (d, C-19), 114.8 (t, C-24), 83.7 (d, C-6), 79.9 (d,C-10), 78.5 (d, C-7), 77.8 (d, C-3), 51.9 (q, CH₃O—), 38.4 (t, C-2),35.6 (t, C-11), 33.6 (t, C-22), 32.2 (t, C-21), 31.6 (t, C-9), 29.6 (t,C-13)*, 29.5 (t, C-14)*, 29.2 (t, C-15)*, 28.9 (t, C-4), 28.0 (t, C-8),27.7 (t, C-16), 26.1 (t, C-12), 25.7 (t, C-5).

[0071] The spectroscopic data of the mixture of stolonoxides A (1) and B(2), in particular the IR absorptions at 3460 cm−¹ and 1745 cm−¹ and the¹³C NMR singlet at δ 175.1 suggested the presence of two isomericcarboxylic acids. Furthermore, the duplication observed in the ¹³C NMRspectrum of the signals attributable to four olefinic methines and twoallylic methylenes, with a relative intensity of 9:1, indicated thatcompounds 1 and 2 only differed in their olefinic pattern. Treatment ofthis mixture with diazomethane afforded the methyl esters 1b and 2b,that could be isolated after repeated HPLC separations.

[0072] The FAB high resolution mass measurement indicated that the majormethyl ester 1b had the molecular formula C₁₅H₄₀O₅ which implies sixdegrees of unsaturation. The presence of the methyl ester group wasconfirmed by the ¹H NMR singlet at δ 3.69 (s, 3H) and the ¹³C NMRsignals at 170.4 (s) and 51.9 (q). The ¹³C NMR spectrum contained sixolefinic carbon signals at δ 138.2 (d), 132.4 (d), 130.8 (d), 124.0 (d),123.4 (d), and 114.7 (t) attributable to a monosubstituted and twodisubstituted double bonds. Furthermore, four doublets of methinecarbons bearing oxygen at δ 83.8 (d), 79.9 (d), 78.5 (d), and 77.6 (d)together with the absence of further carbonyl, methine, or quaternarycarbon signals in the ¹³C NMR spectrum, indicated that 1b was the methylester of a C₂₄ acid containing three double bonds and two oxygenatedrings, which gave rise to the four methines bearing oxygen, andaccounted for the remaining two degrees of unsaturation.

[0073] The absolute stereochemistry of stolonoxide A (1) has beensuggested as 3S, 6S, 7S, 10R by application of the Mosher's method tothe C-3, C-6 diol obtained through peroxide ring opening by catalytichydrogenation. To avoid the apparently confusing results arising fromthe double MTPA esters we employed the Mosher's chiral auxiliary but onthe monohydroxy derivative. This work unambiguously confirms an absolutestereochemistry 3S, 65, 7S, 1OR for stolonoxide A (1).

[0074] The minor methyl ester 2b, obtained by methylation of the naturalmixture of 1 and 2, was isolated as a colorless oil of molecular formulaC₂₅H₄₀O, as indicated by the high resolution mass measurement. A generalinspection of the ¹H and ¹³C NMR of both isomers 1b and 2b, clearlystated that their structures were closely related and that they sharedthe same carbon skeleton and functionalities. However slight differenceswere observed in the signals corresponding to the conjugateddisubstituted double bonds. Thus, the ¹³C NMR spectrum exhibited thesignals of the olefinic carbons at δ 130.5 (d), 128.4 (d), 126.1 (d),and 133.5 (d) which in the HMQC spectrum were correlated with the protonsignals at δ 5.31 (1H, dt, J=11.0 and 7.6 Hz), 5.94 (1H, t, J=11.0 Hz),6.32 (1H, dd, J=15.1 and 11.0 Hz), and 5.65 (1H, dt, J=15.1 and 6.7 Hz),respectively. The coupling constants observed are consistent with adifferent geometry for each of the two conjugated double bonds. Sincethe trains double bond proton signal at δ 5.65 was correlated in theCOSY spectrum with an allylic methylene proton signal at δ 2.20 (2H, m)which was in turn coupled with the signal at 8 2.17 (2H, m) due to themethylene allylic to the terminal double bond, the trans double bondmust be located at C-19, C-20. The ¹³C NMR chemical shifts of theallylic methylene carbons C-21 and C-16 at δ 32.2 (t) and 27.7 (t),respectively, provided confirmation to the proposed ocometry for theC-17, C-18 and C-19, C-20 double bonds. It was characterized thestructure of the methyl ester as 2b and therefore structure 2 wasproposed for stolonoxide B. In the absence of an independentdetermination of the absolute stereochemistry of 2 it was assumed onbiogenetic grounds an identical configuration of the stereogenic centersC-3, C-6, C-7, and C-10 as that determined for stolonoxide A (1).

[0075] It is worth noting that the structure determination of esters 1band 2b aided assignation of the NMR data of the natural mixture ofstolonoxides A (1) and B (2) to each of the individual compounds.

[0076] Careful separation on reversed phase HPLC allowed isolation of amixture of the sodium carboxylate salts 1a and 2a. The inspection of the¹H and ¹³C NMR spectra of 1a and 2a, although clearly indicated thattheir structures were closely related to those of stolonoxides A (1) andB (2), showed some diagnostic differences. Thus, in the ¹³C NMR spectrumthe resonances attributed to C-1, to the C-2 methylene, and to the C-3peroxide methine at δ 177.1 (s), 41.3 (t) and 79.2 (d), respectively,were downfield shifted with respect to the resonances observed incompounds 1 and 2. On the other hand, in the ¹H NMR spectrum the signalsof the methylene protons H-2 and of the methane proton H-3 at δ 2.26(2H, m) and 4.45 (1H, m), respectively, were upfield shifted withrespect to those observed in 1 and 2. These effects can be explained bythe presence in 1a and 2a of a carboxylate group at C-1 instead of thecorresponding carboxylic group, see Kalinowski H. O.; Berger, S.; Braun,S. Carbo-13 NMR Spectroscopy John Wiley and Sons, New York, 1988, pp.198-213, in agreement with the absorption at 1579 cm⁻¹ in the IRspectrum. Treatment of the mixture with HCl and, subsequently, withdiazomethane afforded a mixture of the methyl esters 1b and 2bindicating that 1a and 2a were carboxylate salts of 1 and 2,respectively. As the ESIMS of the mixture 1a and 2a displayed an [M−Na]⁻ion at m/z 405 in the negative mode and the [M+H]⁺ and [M+Na]+ ions at am/z 429 and 451 in the positive mode, it was concluded that thecompounds 1b and 2b isolated from S. socialis were the correspondingsodium carboxylate salts of the stolonoxidc A (1) and B (2).

[0077] The most polar component of the constituents of S. socialis was aminor 6:4 mixture of stolonoxide C and D isolated as their correspondingsodium salts (3 and 4). The IR absorption of 3 and 4 at 1584 cm⁻¹,characteristic of a carboxylate salt, together with the ¹H and ¹³C NMRspectra suggested that the constituents had to be quite similar to thosepresent in the mixture of stolonoxides A and B sodium carboxylates (1aand 2a). The ESIMS ion [M+H]⁺ in the positive mode at m/z 429 wereconsistent with a molecular formula C₂₄H₃₇O₅Na. Treatment of the mixtureof 3 and 4 with HCI and subsequently with diazomethane afforded thecorresponding methyl esters 3b and 4b that unfortunately could not beseparated by HPLC under different conditions. However the structure ofthe methyl esters could be deduced by the spectroscopic study of themixture. Thus, the molecular formula C₂₅H₃₇O₅, obtained from the highresolution mass measurement, indicated that the methyl esters 3b and 4bwere isomers of the stolonoxides A and B methyl esters (1b, 2b).Furthermore, the comparison of the ′H and ¹³C NMR spectra indicated thatthey shared an identical plane structure and that the structuraldifferences had to be due to a different stereochemistry.

[0078] The relative stereochemistry at the stereogenic centers C-3, C-6,C-7, and C-10 was established as follows. The axial orientation of H-3was deduced upon observation of a coupling constant of 11.0 Hz betweenH-3 and H4ax signals and by the correlations exhibited in the ROESYspectrum between the H-3 signal with the H4eq and H-5ax signals whilstthe axial orientation of 14-6 was clear from the observation of acoupling constant of 10.9 Hz between H-5ax and H-6 signals and by thecross peaks observed in the ROESY spectrum between the II-6 signals andthe H-5eq and H-⁴ax signals. Furthermore, the analysis of thecorrelations observed in the ROESY spectrum of the H-7 and H-10 signalswith those of the methylene protons at C-8 and C-9 clearly required H-7and H-10 to be oriented towards opposite sides of the tetrahydrofuranring. However, the stereochemical relationship between both oxygenatedrings could not be unequivocally established by NMR study of the methylesters 3b and 4b. This stereochemical assignment was deduced by acareful study of the unseparable mixture of epoxides 6 and 7 arising bytreatment of the mixture of the methyl esters 3b and 4b with NaOH andsubsequently with diazoniethane. Basically the 1H and ¹³C NMR spectra of6 and 7 were quite similar to those of the epoxide 5 discussed aboveexcepting for the ¹H NMR signal of the H-6 at δ 3.79 (1H, m) and the ¹³CNMR doublet of C-6 at δ 71.3. These chemical shifts fit better for anerythro orientation of the substituents around C-6 and C-7 stereogeniccenters rather than the alternative threo orientation present in epoxide5, see Alai, F. Q.; Liu, X. X.; McLaughlin, J. L. J. Nat. Prod. 1999,62, 504-540. Based in this result it was proposed the relativestereochemistry 3S*, 6S*, 7R*, 1OS* for the stolonoxides C and D sodiumsalts (3a and 4a).

1. A pharmaceutical composition comprising a stolonoxide compound and apharmaceutically acceptable carrier.
 2. A pharmaceutical compositionaccording to claim 1, where the stolonoxide compound is of the formula:

or a derivative thereof.
 3. A pharmacutical composition according toclaim 2, where the stolonoxide compound is stolonoxide A, B, C or D ofthe respective formula:

where R is H, or a derivative of one of these compounds.
 4. A method oftreating a mammal affected by cancer which comprises administering tothe affected individual a therapeutically effective amount of astolonoxide compound, or a pharmaceutical composition thereof.
 5. Theuse of a stolonoxide comound in the preparation of a pharmaceuticalcomposition for the treatment of a tumour.
 6. A stolonoxide compound,with the exception of the known methyl ester of stolonoxide A offormula:


7. A stolonoxide compound according to claim 6, where the compound isstolonoxide A, B, C or D of the formulae:

where R is H for the parent stolonoxide compound, or a derivativethereof where R is not hydrogen.